Our Unit is interested in the molecular mechanisms underlying regulated patterns of gene expression, both in time and space. During FY 00 we have extended our studies of the contextual properties of circadian E-boxes. This work is important because the E-box is an extremely promiscuous DNA consensus element; thus, in order to understand how different signaling pathways can differentiate among different E-boxes, a systematic analysis of prototypic promoters is needed. By subjecting the cyclin B1 (CYC), vasopressin (AVP) and arylalkylamine N-acetyltransferase (NAT) E-box-containing promoters to such an analysis, we have obtained significant new information regarding the modular nature of these regulatory cassettes and how they are put together in order to achieve specificity. In a related collaborative study we have established two new lines of transgenic rats carrying a luciferase reporter gene driven by either the wild type or an E-box-mutated NAT promoter. We believe that these transgenic lines will help us test our hypothesis that the NAT E-box plays an important role in the regulation of NAT in the master oscillator cells of the retina. In contrast, we predict that pineal control of NAT, which revolves around cAMP responsive elements, will prove refractory to E-box inactivation. A second project is derived from our discovery during FY 99 of a novel gene, using a yeast two hybrid system, that appears to interact specifically with Period (Per) 1, one of the core components of the cellular machinery that generates the self-sustained 24 hr cycle in transcriptional output. We have now developed an excellent antiserum directed against this novel gene product, which allowed us to establish that the protein is expressed exclusively in neurons. The knockout approach to determine the function of this gene in vivo is still underway.